With the advent of artificial reproductive technologies such as in-vitro fertilization which involves follicular aspiration, cryo-preservation, etc., there is a need for precisely extracting oocytes by separating the oocytes from body fluids such as blood, follicular fluid, etc. Follicular aspiration is a procedure that extracts oocytes from the follicles, for example, by puncturing the follicles in a human ovary with a needle which is introduced through the vagina and guided inside the follicles using ultrasound vision. An oocyte is a female gametocyte or a germ cell involved in reproduction. That is, the oocyte is an immature ovum, or an egg cell.
Primary oocytes are present in a human ovary at birth; thereafter, a human female does not form any more oocytes throughout her lifetime. Once a woman reaches sexual maturity, the woman typically starts to ovulate a single oocyte each cycle. The oocytes contained within the ovary are surrounded by a tightly packed corona and granulosa cells. The granulosa cells are also referred to as cumulus cells. The oocyte, the corona, and the granulosa cells are typically referred to as a cumulus complex. One or more oocytes start to mature each month. This results in a development of a fluid filled sac called a follicle around the oocyte and the granulosa or the cumulus complex. As the oocyte matures, the cells become more loosely packed and the size of the follicle increases around the oocyte, until eventually the follicle ruptures. The rupture of the follicle and subsequent release of the oocyte and other follicular contents is referred to as ovulation.
A practical application of culturing of oocytes is in-vitro fertilization. The process of in-vitro fertilization (IVF) requires that the oocytes be collected prior to the occurrence of ovulation. An aspiration needle is used to penetrate each follicle in the ovary using, for example, ultrasound guidance or laparoscopy. The contents of each follicle comprising follicular fluid, oocytes surrounded by corona cells and granulosa cells, detached granulosa cells and corona cells, blood comprising a mixture of red blood cells, white blood cells, plasma, etc., are aspirated into a test tube. As the aspirated fluid mixture passes into the test tube, the fluid mixture is observed, usually by a medical assistant such as a nurse, who informs a medical practitioner to stop the process of aspiration once the level of the fluid mixture approaches an outlet tube. A stopper initially inserted in the test tube is then removed from the test tube and inserted into a fresh test tube into which the next batch of follicular contents is to be transferred. The medical practitioner can then flush or rinse out the collapsed follicle with a flushing fluid which is also aspirated into a test tube. The medical practitioner may then aspirate and flush any other follicles which may be present using the flushing fluid. The flushing fluid is a fluid used for irrigating or rinsing out the collapsed follicles after the contents of the follicles are extracted, or for cleansing the contents in the test tube, by a rapid flow of the fluid. The flushing fluid is, for example, a saline solution, culture media, or other solutions of various salts designed to closely match the fluid found within human fallopian tubes. Each time the fluid level of the fluid mixture in the test tube approaches the outlet tube, the medical practitioner must pause to allow the medical assistant to change the test tube. The filled test tubes are then passed to an embryologist who transfers the contents into a Petri dish and thereafter attempts to locate and isolate any oocytes from the transferred fluid mixture.
Some medical practitioners prefer to keep the contents of each follicle, that is, the oocytes, follicular fluid, oocytes surrounded by corona cells and granulosa cells, detached granulosa cells and corona cells, blood comprising a mixture of red blood cells, white blood cells, plasma, etc., separate while other medical practitioners prefer to group the contents together. The medical practitioner may continue to aspirate follicles until there are no more follicles above a certain size remaining to aspirate. The aspiration process is slowed by having to transfer the stopper into a fresh test tube as the test tube fills up before passing the test tube to an embryologist. This may need constant supervision from a medical assistant who needs to continually monitor the rise of the fluid level in the test tube and to inform the medical practitioner to stop aspirating to allow the stopper to be removed and then inserted into the next fresh test tube. This process may need to be performed several times for each follicle.
Conventional methods and apparatuses for oocyte separation and collection often require that the oocytes be meticulously extracted by skilled clinical technicians using macroscopic and microscopic examination of a combined mixture of fluids comprising, for example, blood fluid, follicular fluid, flushing fluid, etc. Furthermore, the examination and subsequent extraction of oocytes using conventional methods of oocyte separation and collection is often difficult since the waste fluids often comprise blood, which is prone to clotting. Therefore, these methods are also often time consuming and increase the complexity of isolation of oocytes.
Furthermore, conventional methods of oocyte separation and collection often expose the oocytes to temperature variations that may expose the oocytes to thermal stress. When oocytes are collected by a conventional method, the oocytes move from the environment of the human body at approximately 37 Celsius to a room temperature environment of approximately 20 Celsius which is a considerable drop in the surrounding ambient temperature. While suitable efforts are made during the isolation process to maintain the oocytes in their original environmental conditions, that is, according to the environment of the human reproductive system, when the oocytes pass through a fine tubing of an aspiration assembly, the oocytes are not heated and not maintained at a temperature of the environment of the human reproductive system. A test tube that collects the oocytes is typically positioned in a heated test tube rack where the test tube is surrounded by heat conducing material from all sides except the top. However, when the fluid mixture containing the oocytes is poured into a Petri dish for identification and separation of the oocytes from the fluid mixture in the conventional method, there may be a drop in temperature and also exposure to air. The Petri dish is usually examined on a heated microscope stage but the heat only comes, for example, from a base heating element positioned below the Petri dish. These temperature variations induced in the conventional isolation method may expose the oocytes to thermal stress which may interfere with the viability of maintaining the oocytes in a culturing system.
Moreover, conventional methods are often vulnerable to the risk of clotting of the fluids due to exposure of the blood in the fluids to air while aspirating a follicle. Furthermore, in conventional methods for separation and collection of oocytes, oocytes need to be meticulously extracted from a combined mixture of oocytes and associated waste fluids from a Petri dish. However, there is a possibility that the oocytes could be damaged due to variations in the temperature of the Petri dish, changes in potential hydrogen (pH) levels, osmolarity, etc., while transferring the oocytes to the Petri dish or from the Petri dish, etc.
Furthermore, the fluids extracted from the follicle and the flushing fluid for cleaning the oocytes are often injected through an inlet tube that directs the extracted fluids and the flushing fluid into a test tube in a free fall motion which allows the formation of air bubbles within a conventional oocyte collection apparatus. The formation of air bubbles, for example, may obscure the macroscopic and microscopic visualization of the oocytes. Furthermore, the oocytes may be exposed to mechanical stress as a result of the free fall motion of the fluids containing the oocytes and the flushing fluid, in the conventional oocyte collection apparatus, potentially damaging the oocytes.
The oocytes are generally collected sequentially, one at a time unless a medical practitioner penetrates two or more follicles simultaneously. The contents from each follicle may be divided between two or more test tubes, each of which must be meticulously searched by an embryologist for oocytes. There is a need for an apparatus that collects only the desirable contents of a follicle or more than one follicle in a single test tube and passes the test tube to the embryologist for considerably speeding up the work of the embryologist and hence the entire procedure.
Hence, there is a long felt but unresolved need for an apparatus and a method that quickly performs separation and collection of a single oocyte or multiple oocytes at a time by filtering a fluid mixture comprising one or more oocytes and extraneous fluids, while minimizing exposure of the oocytes to thermal stress and mechanical stress, without causing damage to the oocytes, thereby collecting oocytes that are more easily contained, harvested, and available for storage and use. Furthermore, there is a need for an apparatus and a method that enables transfer of only the extraneous fluids of the fluid mixture out of an oocyte receptacle, thereby allowing retention and collection of only the oocytes in a minimal volume of the fluid mixture in the oocyte receptacle. Furthermore, there is a need for an apparatus and a method that enables medical practitioners to contain the oocytes and therefore preclude the risk of inadvertently discarding the oocytes.